Clonal cell lines (BME-UV) were established from primary epithelial cells by stable transfection with a plasmid, carrying the sequence of the simian virus 40 early region mutant tsA58, encoding the thermolabile large T antigen. The BME-UV cells have undergone more than 300 population doublings and produce intranuclear large T antigen. At low confluency, growing islands of cells are apparent exhibiting the characteristic cobblestone morphology of epithelial cells. The BME-UV cells expressed functional markers such as microvilli and desmosomes and biochemical markers of mammary epithelial cells such as a repertoire of cytokeratins. The BME-UV cells are capable of synthesizing low levels of α-lactalbumin and α_8l (50 ng/ml of medium/24 h). One of the cell lines, BME-UV1 showed enhanced proliferation in the presence of epidermal growth factor (EGF) and insulinlike growth factor I (IGF-I). The BME-UV1 cell line is the only known bovine mammary epithelial cell line responsive to EGF. The BME-UV cells grown on collagen at low confluency are capable of developing very long projections that most likely allow for communication between cells at a distance from each other. The BME-UV cells may become a valid model system to examine bovine mammary epithelial proliferation and differentiation and cell-to-cell communication.